Project Summary/Abstract The goal of this project is to complete the pre-clinical and translational studies necessary to prepare a novel oncolytic viral (OV) therapy, WO-S3, for clinical evaluation in non-small cell lung cancer (NSCLC). NSCLC is the predominate form of lung cancer, which is the leading cause of cancer death in the United States. Recently, immunotherapies have emerged as a potential new treatment approach in NSCLC but have been limited by low response rates and designated primarily for use in second-line treatment. However, the ability of OVs to exploit the immune system has been demonstrated in other solid tumor types, such as melanoma, and so they hold great promise for use in NSCLC, especially if effective systemic delivery were possible. Oncolytic viruses have the potential to incorporate multiple features into a single viral backbone, to target cancer cells directly (to induce cell death), to overcome localized immune suppression within the tumor, and to boost the anti-tumor adaptive immune response while evading anti-viral immune responses. To date, no effective OV therapies have been developed for use in NSCLC, primarily due to the need to delivery most OVs directly into the tumor, a delivery mechanism that is often not feasible in lung cancer. WO-S3 represents a next generation of OV therapies, and has been engineered to contain multiple genetic modifications and expresses several transgenes, which, collectively, overcome many of the limitations facing previous generations of OVs. Modifications in WO-S3 allow for greatly enhanced intravenous administration, increased tumor-selective viral replication, as well as targeting of activated STAT3, a protein that is a key driver of many of the hallmarks of cancer. Furthermore, WO-S3 overcomes immune suppression in the tumor. Previous pre-clinical work has demonstrated WO-S3's mechanism of action and potency against tumors, including mouse models of lung cancer. We believe that this therapy has significant potential to advance the treatment of NSCLC, and advancing WO-S3 to clinical testing is of importance. The work described in this current proposal is specifically aimed at completing the studies necessary to prepare for clinical evaluation. As such, we will demonstrate that our existing manufacture process can be used to create WO-S3 at clinical grade and in sufficient quantities for use in clinical testing, with the ultimate goal of producing an economically viable product. In addition, we will complete the FDA-mandated safety and toxicology testing through a series of mouse studies. We will develop all assays necessary to confirm the clinical product (WO-S3) is as intended, and these will be transferred to a contract manufacturing facility where a viral bank of WO-S3 will be created for use as seed material in all future manufacturing runs for clinical testing as well as eventual commercial use. In addition, mouse studies will be run to define the optimal treatment regimen for the use of WO-S3 alone or in combination with approved immunotherapies in the treatment of NSCLC. As a result, first in man studies will be possible soon after the completion of this proposal.